WO2024004981A1

WO2024004981A1 - Collagen-rich organic composition and production method thereof

Info

Publication number: WO2024004981A1
Application number: PCT/JP2023/023721
Authority: WO
Inventors: 直登大久保; 秀崇細野
Original assignee: 株式会社DeVine
Priority date: 2022-06-27
Filing date: 2023-06-27
Publication date: 2024-01-04

Abstract

[Problem] To provide: a collagen-rich organic composition; a biomaterial using the collagen-rich organic composition as a starting material; a biomaterial or other products containing collagen as a main ingredient; a method for producing the collagen-rich organic composition; a method for differentiating a tooth or a bone extracted from a mammal as a starting material for a biomaterial or a biomaterial or other products containing collagen as a main ingredient; and a method for assessing the potential risk of a disease in a mammal and/or whether the mammal is potentially in good health or not with the use of PYD and PYD and PpP as indicators. [Solution] PYD>100, and PYD>100 and PpP>740.

Description

Collagen-rich organic composition and method for producing the same

The present invention relates to a collagen-rich organic composition produced from teeth or bones extracted from a mammal, a biomaterial made from the collagen-rich organic composition as a raw material, a biomaterial or other product containing collagen as a main component, A method for producing the collagen-rich organic composition, a method for identifying teeth or bones extracted from a mammal as a raw material for biomaterials, biomaterials containing collagen as a main component, or other products, and mammals. Quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth (ng/mg) as an index for evaluating the potential risk of disease and/or whether the mammal is potentially healthy. , or a quantitative value of the amount (ng/mg) of pyridinoline contained in collagen contained in mammalian teeth, and a quantitative value of the amount (ng/mg) of pyridinoline contained in collagen contained in mammalian teeth, and The present invention relates to a method using a weight ratio (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) calculated from a quantitative value of the amount of pentosidine (ng/mg).

Conventionally, various collagens (actually, "collagen-rich organic composition" is more accurate, but in general, they are often simply referred to as "collagen") have been developed. Most of them are atelocollagen produced by extraction from soft tissue in living bodies using an enzyme treatment method, and the telopeptides present at the N-terminus and C-terminus of the amino acid sequence of collagen are cleaved, and , collagen from which poorly soluble collagen fibers have been removed through the purification process. In addition, materials extracted from hard tissues such as bones and teeth, such as demineralized Freeze-Dried Bone Allograft (DFDBA) and demineralized dentin matrix (DDM), are also available. Collagen produced by Collagen is produced through demineralization and is mainly used for biomaterials.

The quality of atelocollagen is evaluated because the telopeptide, which is said to have antigenic properties, has been cleaved by enzyme treatment. The quality of collagen (which has a triple helical structure due to the fact that it has a triple helical structure) is evaluated based on how highly pure collagen is produced by setting the purification treatment conditions such as pH. By the way, when producing collagen from hard tissue, it is necessary to remove minerals and extract collagen by demineralization treatment, that is, acid treatment, which limits the yield of collagen and makes extraction time-consuming. Therefore, almost all collagen products that have been developed are soft tissue-derived atelocollagen, undenatured collagen, or products using them.

Most of the collagen contained in living tissues is type I collagen (type I collagen). Type I collagen has physiological cross-links and non-physiological cross-links; the physiological cross-link molecules are pyridinoline cross-link molecules, and the non-physiological cross-link molecules are advanced glycation end products (AGEs), typified by pentosidine. ) are known (Non-Patent Document 1). Pyridinoline crosslinking is a physiological/enzymatic crosslinking caused by the action of lysyl oxidase, and as it is a regular crosslinking molecule at a specific site in the amino acid sequence of collagen, it contributes to improving the elasticity and strength of collagen fibers. ing. On the other hand, pentosidine cross-linking is a non-physiological, non-enzymatic cross-linking involving blood sugar (aging cross-linking (AGEs cross-linking)), and when blood sugar is warmed by body temperature, arginine and lysine dotted in the amino acid sequence of collagen Because it is a crosslinking molecule formed between arginine and lysine that are close to each other in the amino acid sequence of collagen, it is a crosslinking molecule that is randomly formed between adjacent arginine and lysine in the amino acid sequence of collagen, and is a factor that disturbs the physiological three-dimensional structure of collagen. This is caused by a decrease in fiber elasticity and strength. As mentioned above, atelocollagen not only contains almost no pyridinoline crosslinked molecules because the telopeptide has been cleaved by enzymatic treatment, but also because the purification process eliminates poorly soluble collagen fibers caused by pentosidine crosslinking. , is a collagen that also contains almost no pentosidine cross-linked molecules.

It has been known that an increase in the number of pyridinoline cross-linked molecules in collagen increases the strength and elasticity of the collagen (Non-Patent Document 2), and it is also known that pentosidine cross-links are aging cross-links (AGEs cross-links). (Non-patent Document 3). Furthermore, the amount of pentosidine per unit collagen in human articular cartilage increases linearly with age, and the ratio of the amount of pentosidine to the amount of pyridinoline per unit collagen (amount of pentosidine contained/amount of pyridinoline contained) increases with age. It has been reported that the ratio between the amount of pentosidine contained and the amount of pyridinoline contained per unit collagen (amount of pentosidine contained/pyridinoline contained There is a report that the amount) increases significantly (Non-Patent Document 6).

The above-mentioned evaluation of the quality of collagen (organic composition with high collagen content) is generally evaluated based on the purity and origin of collagen in the organic composition with high collagen content, and some molecular level indicators of collagen are evaluated. In other words, it is not evaluated based on any index at the amino acid sequence level of collagen, so it is difficult to say that the quality of collagen itself is accurately evaluated. The amount of pyridinoline contained, which contributes to improving the quality of collagen, and the amount of pentosidine contained, which is a factor that disturbs the physiological three-dimensional structure of collagen, resulting in a decrease in the elasticity and strength of collagen fibers, which in turn contributes to a decrease in the quality of collagen. The quality of collagen has not been evaluated based on the amount or the weight ratio of the amount of pentosidine contained and the amount of pyridinoline contained and the amount of pentosidine contained.

In addition, conventionally, collagen-rich organic compositions and collagen-based compositions have been evaluated as essentially containing high-quality collagen based on the molecular level of some collagen, that is, the amino acid sequence level of some collagen. Not only have we not found any biomaterials that can do this, but also the telopeptides mentioned above remain, which limits the yield of collagen, and it is difficult to extract collagen from hard tissues, especially collagen, which is difficult to extract and has a low yield. For teeth, which are limited in number, and bones, which require a great deal of effort to extract collagen because they contain large amounts of lipids, it is possible to determine whether the quality is essentially good based on some kind of collagen molecular level, that is, some kind of collagen amino acid sequence level. No attempt has been made to provide a collagen-rich organic composition that can be evaluated as containing collagen or a biomaterial containing collagen as a main component.

By the way, the telopeptide region of collagen is thought to exhibit high antigenicity, so some collagen products use atelocollagen from which telopeptides have been removed through enzyme treatment; As a result, the pyridinoline crosslinking molecules are also lost, and the flexibility and toughness characteristic of collagen are lost. Furthermore, there is no clear evidence that removing telopeptides reduces antigenicity, and therefore, in medical applications of collagen, removing telopeptides has no benefit on immunogenicity. On the other hand, the percentage of humans who are allergic to bovine type I collagen is 2-4%, which is equivalent to a nickel hypersensitivity rate of 10-15% and a latex allergy rate of approximately 6%. (Lynn AK et al., J Biomed Mater Res B Appl Biomater. 2004 Nov. 15; 71 (2): 343-354). It should be noted that there are reports that type I collagen does not cause an immune reaction or does not show antigenicity (Courtenay JS et al., Nature, 283, 14, 1980, 666-668; David E et al., J. Exp. Med. 1977, 46; 857-868).

In addition, in the above-mentioned non-patent documents 1 to 6, there are certainly various references to pyridinoline crosslinked molecules and pentosidine crosslinked molecules in collagen, and the ratio of the amount of pentosidine contained per unit collagen to the amount of pyridinoline contained in diseases. (Amount of pentosidine contained/amount of pyridinoline contained) is discussed, but none of the documents specifically deals with collagen derived from teeth, which is not easy to extract. In addition, in tissues that physiologically require strength, such as hard tissues such as bones and cartilage, and soft tissues such as tendons and ligaments, the amount of pyridinoline cross-linked molecules contained in collagen and the corresponding amount of pentosidine cross-linked molecules are large. (M. TAKAHASHI et al., Anal Biochem Vol. 232, 158-62, 1995), but since these tissues undergo metabolism, the pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained therein also Because it is metabolized, it does not reflect the amount of pyridinoline cross-linked molecules or pentosidine cross-linked molecules accumulated over the lifetime of the mammalian host. On the other hand, teeth are hard tissues that are not incorporated into the metabolic cycle, and therefore the pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained in the collagen contained therein are not metabolized. This reflects the accumulated amount of pyridinoline cross-linked molecules and pentosidine cross-linked molecules for almost the entire lifetime of the mammalian host. Therefore, in order to accurately assess the potential risk of disease in a mammal and whether the mammal is potentially healthy, it is necessary to obtain a lifetime supply of pyridinoline- and pentosidine-crosslinked molecules from the mammalian host. It is clear that accumulated tooth-derived collagen (organic composition with high collagen content) should be used as an indicator. Furthermore, the collagen-rich organic composition and collagen-based biomaterial according to the present invention are subjected to strong alkaline treatment, which is not normally applied to collagen-containing proteins, at temperatures higher than room temperature, resulting in desorption. Because it is manufactured through ash treatment, i.e., acid treatment, the amount of endotoxin it contains is considerably reduced, and it is essentially free of endotoxin, or the endotoxin it contains is substantially inactivated. However, in the above-mentioned non-patent documents 1 to 6, there is no such description, let alone any suggestion.

The present invention is a quantitative value of the amount of pyridinoline (ng/mg) contained in collagen and the amount of pentosidine (ng/mg) contained in collagen, which is determined using a predetermined method. From PYD and PEN, which is the quantitative value of the amount of pentosidine contained (ng/mg), PpP, which is the value of the weight ratio (amount of pyridinoline contained (ng/mg) / amount of pentosidine contained (ng/mg)), is calculated, A collagen-rich organic composition produced from a tooth or bone extracted from a mammal, which has the predetermined PYD or the PYD and the PpP, and a biological material using the collagen-rich organic composition as a raw material, collagen. To provide a biomaterial or other product as a main component, and a method for producing the collagen-rich organic composition, when the PYD or the PYD and the PpP are predetermined values, the tooth or bone is From mammals, which is a raw material for biomaterials, collagen-based biomaterials or products, characterized by having a step of selecting the material as a raw material for biomaterials, collagen-based biomaterials, or other products. Pyridinoline content of collagen contained in mammalian teeth, which is a method for identifying extracted teeth or bones, evaluating the potential risk of disease and/or whether the mammal is potentially healthy. PYD, which is a quantitative value of the amount (ng/mg), and PEN, which is a quantitative value of the amount of pentosidine contained (ng/mg), are obtained, and from the obtained PYD and the obtained PEN, the weight ratio value is obtained. A step of calculating a certain PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)), and a mammal not suffering from a disease, a mammal suffering from a disease, and a healthy mammal. and R-PYD, which is a quantitative value of the amount of pyridinoline (ng/mg) contained in collagen contained in the teeth of one or more mammals selected from unhealthy mammals, and the amount of pentosidine contained as a reference. Obtain R-PEN, which is a quantitative value of the amount (ng/mg), and calculate R-PpP (containing A step of calculating the amount of pyridinoline (ng/mg)/amount of pentosidine contained (ng/mg)) and comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD, the PpP and the As an indicator for evaluating the potential risk of disease in a mammal and/or whether or not the mammal is potentially healthy, the method comprises a step of comparing R-PpP. Quantitative value of the amount of pyridinoline contained in collagen (ng/mg) contained in teeth, or quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth (ng/mg), and quantitative value of pyridinoline content (ng/mg) contained in collagen contained in mammalian teeth. Weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen (ng/mg) and the amount of pentosidine contained (ng/mg) (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) The purpose is to provide a method for using.

As a result of extensive research in order to solve the above problems, the present inventors have developed the method of applying crushed teeth or bones extracted from mammals to a strong alkali under temperature conditions higher than room temperature and lower than the boiling point of water. Collagen-rich organic composition obtained by decalcifying the strongly alkali-treated pulverized product under negative pressure conditions, biomaterials produced from biomaterials, biomaterials containing collagen as a main component, or other products. PYD, which is the quantitative value of the amount of pyridinoline contained per unit collagen (ng/mg), and the weight ratio calculated from the amount of pyridinoline contained per unit collagen (ng/mg) and the amount of pentosidine contained (ng/mg). When the value PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) is a predetermined value, the collagen contained has a three-dimensional structure that provides excellent elasticity and strength. , is of good quality, and when the PYD or the PYD and the PpP are at predetermined values, the material is extracted from a mammal and serves as a raw material for biomaterials, collagen-based biomaterials, or other products. Being able to identify teeth or bones, collagen contained in the teeth of a mammal that is subject to evaluation of the potential risk of contracting a disease and/or whether or not the mammal is potentially healthy; The above-mentioned PYD for each of the collagens contained in the teeth of one or more mammals selected from non-healthy mammals, diseased mammals, healthy mammals, and unhealthy mammals. The inventors discovered that by comparing PYD and PpP, it is possible to evaluate the potential risk of disease in a mammal and/or whether or not the mammal is potentially healthy, and completed the following inventions. .

(1) A collagen-rich organic composition manufactured from teeth or bones extracted from a mammal, which is the following (i) or (i) and (ii);
(i) The amount of pyridinoline contained in collagen contained in the collagen-rich organic composition (ng/ PYD, which is a quantitative value of mg), is PYD>100,
(ii) The amount of pyridinoline contained in the collagen contained in the collagen-rich organic composition (ng/ PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg) ), PpP>740.

(2) The collagen according to (1), which does not substantially contain endotoxin, or contains endotoxin, or contains substantially inactivated endotoxin, or has endotoxin inactivated. High content organic composition.

(3) The collagen-rich organic material according to (1), wherein the mammal is one or more mammals selected from the group consisting of cows, pigs, horses, sheep, deer, dogs, cats, and humans. Composition.

(4) A biomaterial made from the collagen-rich organic composition described in any one of (1) to (3), a biomaterial containing collagen as a main component, or other products.

(5) obtaining a crushed product by crushing teeth or bones extracted from a mammal;
a step of treating the obtained pulverized material with a strong alkali;
The method for producing a collagen-rich organic composition according to any one of (1) to (3), which comprises a step of decalcifying the pulverized product treated with a strong alkali under negative pressure conditions.

(6) The step of treating the obtained pulverized material with a strong alkali is a step of treating the obtained pulverized material with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water. ).

(7) quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in teeth or bones extracted from mammals;
Quantifying the amount of pentosidine (ng/mg) contained in collagen contained in teeth or bones extracted from the mammal;
PpP (the amount of pyridinoline contained (ng /mg)/amount of pentosidine contained (ng/mg));
PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) or PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) and PpP (the amount of pyridinoline contained (ng /mg)/pentosidine content (ng/mg)) is a predetermined value, the teeth or bones extracted from the mammal can be used as biomaterials, collagen-based biomaterials, or raw materials for other products. A method for identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a collagen-based biomaterial, or other products, comprising:

(8) obtaining a crushed product by crushing teeth or bones extracted from a mammal;
a step of treating the obtained pulverized material with a strong alkali;
Deashing the strong alkali-treated pulverized material under negative pressure conditions to obtain an organic composition with a high collagen content;
Quantifying the amount of pyridinoline contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition;
Quantifying the amount of pentosidine contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition;
PpP (the amount of pyridinoline contained (ng /mg)/amount of pentosidine contained (ng/mg));
PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) or PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) and PpP (the amount of pyridinoline contained (ng /mg)/pentosidine content (ng/mg)) is a predetermined value, the teeth or bones extracted from the mammal can be used as biomaterials, collagen-based biomaterials, or raw materials for other products. A method for identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a collagen-based biomaterial, or other products, comprising:

(9) Quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in the obtained organic composition with high collagen content and the amount of pentosidine contained in collagen contained in the obtained organic composition with high collagen content ( ng/mg) contained in the obtained collagen-rich organic composition by using a high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. A step of quantifying the amount of pyridinoline contained in collagen (ng/mg) and a step of quantifying the amount of pentosidine contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition, and The method according to (7) or (8), wherein the values are PYD>100 or PYD>100 and PpP>740, respectively.

(10) Quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth as an indicator for evaluating the potential risk of disease in mammals and/or whether or not the mammal is potentially healthy. , or the weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the quantitative value of the amount of pentosidine contained in collagen. A method to use,
Quantifying and quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in the teeth of a mammal, which is the target of evaluating the potential risk of disease and/or whether or not the mammal is potentially healthy. Obtaining the value PYD;
A step of quantifying the amount of pentosidine (ng/mg) contained in the collagen contained in the teeth of the mammal to be evaluated to obtain a quantitative value of PEN;
A step of calculating a weight ratio value of PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) from the obtained PYD and the obtained PEN;
The amount of pyridinoline contained in collagen contained in the teeth of one or more mammals selected from a mammal not suffering from a disease, a mammal suffering from a disease, a healthy mammal, and an unhealthy mammal. (ng/mg) to obtain a reference quantitative value of R-PYD;
The amount of pentosidine contained in collagen contained in the teeth of one or more mammals selected from a mammal not suffering from a disease, a mammal suffering from a disease, a healthy mammal, and an unhealthy mammal. (ng/mg) to obtain R-PEN, which is a reference quantitative value;
From the obtained R-PYD and the obtained R-PEN, calculate the reference value of weight ratio R-PpP (contained pyridinoline amount (ng / mg) / contained amount of pentosidine (ng / mg)) The process of
Comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and the PpP and the R-PpP.

(11) A step of quantifying the amount of pyridinoline contained (ng/mg) to obtain R-PYD, which is a reference quantitative value, and quantifying the amount of pentosidine contained (ng/mg), and obtaining R-PYD, which is a reference quantitative value. The step of obtaining PEN is to quantify the amount of pyridinoline contained (ng/mg) using a high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents to obtain a reference quantitative value. A step of obtaining R-PYD and a step of quantifying the amount of pentosidine contained (ng/mg) to obtain R-PEN as a reference quantitative value, and comparing the PYD and the R-PYD, or The step of comparing the PYD and the R-PYD and comparing the PpP and the R-PpP confirms whether the PYD is PYD>100, or the PYD is PYD>100. The method according to (10), which is a step of confirming whether or not PpP is greater than 740.

According to the present invention, a crushed tooth or bone extracted from a mammal is treated with a strong alkali (preferably treated with a strong alkali under predetermined conditions such as a temperature higher than room temperature), and the strong alkali treatment is performed. A high-quality collagen-rich organic composition, which is obtained by decalcifying the pulverized material under negative pressure conditions, has a three-dimensional structure that provides excellent elasticity and strength, and biomaterials and collagen. It is possible to provide biomaterials or other products based on collagen, and to identify teeth or bones extracted from mammals as raw materials for biomaterials, collagen-based biomaterials, or other products. Furthermore, the content of pyridinoline in collagen contained in mammalian teeth can be used as an indicator for evaluating the potential risk of disease in the mammal and/or whether the mammal is potentially healthy. Quantitative value of pyridinoline content (ng/mg), or quantitative value of pyridinoline content (ng/mg) of collagen contained in mammalian teeth, and quantitative value of pyridinoline content (ng/mg) of collagen contained in mammalian teeth. The weight ratio (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) calculated from the quantitative value of the amount of pentosidine (mg) and the amount of pentosidine contained (ng/mg) can be used.

18 cases in humans, 36 cases in cattle (including 19 cases in bovine Specified Risk Materials (SRM), 17 cases in Tokachi young cattle (registered trademark in Japan, early fattening (14 months old) male Holstein); A high-speed liquid with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pairing reagents of collagen contained in an organic composition with high collagen content derived from dentin of 8 teeth of domestic pigs (6 months old) (62 cases in total). FIG. 2 is a graph showing the distribution of PYD, which is the quantitative value of the amount of pyridinoline contained (ng/mg), determined by chromatography (HPLC-Flu). Organic composition with a high content of collagen derived from dentin from the teeth of 18 humans, 36 cows (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs (62 cases in total) Collagen contained in the product (n=62), Telplug (n=3, atherized collagen derived from bovine dermis, telopeptide-free treatment), high-grade gelatin (n=3, porcine acid treatment, telopeptide preservation type treatment) method, low endotoxin collagen), heptafluorobutyric acid and formic acid in the gingiva (n = 3) of Tokachi Young Cattle (registered trademark in Japan), and the muscle (n = 3) of Tokachi Young Cattle (registered trademark in Japan). FIG. 2 is a graph showing the distribution of PYD, which is the quantitative value of the amount of pyridinoline contained (ng/mg), determined by a high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) used as an ion pairing reagent. In the figure, the broken line indicates the PYD threshold. Organic composition with high content of collagen derived from dentin from teeth of 18 humans, 36 cows (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs (62 cases in total) PEN is the quantitative value of the amount of pentosidine contained in collagen (ng/mg) determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. It is a graph diagram showing the distribution of. Organic composition with a high content of collagen derived from dentin from the teeth of 18 humans, 36 cows (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs (62 cases in total) PYD is the quantitative value of the amount of pyridinoline (ng/mg) contained in collagen, determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. Distribution of values of PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg)), which is the value of the weight ratio calculated from PEN, which is the quantitative value of the contained pentosidine amount (ng/mg) FIG. Endotoxin test (LAL method) of collagen contained in an organic composition with high collagen content derived from the dentin of teeth of 6 Tokachi Wakagyu cows (registered trademark in Japan), 3 cases of gelatin derived from pig skin, and 3 cases of high-grade gelatin FIG. 2 is a graph showing the distribution of endotoxin values (endotoxin level; EU/mg) quantified by.

Hereinafter, a collagen-rich organic composition produced from a tooth or bone extracted from a mammal, a biomaterial made from the collagen-rich organic composition, a biomaterial containing collagen as a main component, or other materials according to the present invention will be described below. A method for producing the collagen-rich organic composition, a method for identifying teeth or bones extracted from a mammal as a raw material for biomaterials, biomaterials containing collagen as a main component, or other products, and , the amount of pyridinoline contained in collagen contained in the teeth of the mammal (ng/mg ), or the quantitative value of the amount (ng/mg) of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount (ng/mg) of pyridinoline contained in collagen contained in mammalian teeth. A method using a weight ratio (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) calculated from a quantitative value of the amount of pentosidine contained (ng/mg) will be described in detail. In this specification, a numerical range expressed using "~" or "from" means a range that includes the numerical values written before and after "~" or "from" as lower and upper limits.

The collagen-rich organic composition according to the present invention is a collagen-rich organic composition manufactured from teeth or bones extracted from mammals, and the collagen is the following (i) or (i) and (ii). It is a high content organic composition.
(i) The amount of pyridinoline contained in collagen contained in the collagen-rich organic composition (ng/ PYD, which is a quantitative value of mg), is PYD>100,
(ii) The amount of pyridinoline contained in the collagen contained in the collagen-rich organic composition (ng/ PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg) ) is PpP>740.

In the present invention, the term "mammal" refers to an animal that basically reproduces sexually, and many of the existing species are viviparous and raise their young with milk, and there are no particular limitations on such animals. Such animals include, for example, humans; apes such as orangutans, gorillas, chimpanzees, bonobos, and monkeys belonging to the family Gibbonidae; monkeys other than monkeys belonging to the family Gibbonidae, as well as cows, horses, pigs, wild boars, sheep, and goats. , large mammals such as antelopes, bears, sea lions, seals, walruses, sea lions, fur seals, and whales; small mammals such as dogs, cats, rabbits, rats, squirrels, weasels, raccoons, and mongooses; Cows, pigs, horses, sheep, deer, dogs, cats, humans can be mentioned as preferred mammals, cows, pigs and horses can be mentioned as more preferred mammals, cows and pigs are the most preferred mammals. It can be mentioned as a mammal.

"Collagen" is one of the proteins that forms the framework of various tissues in living organisms, mainly constituting various organs, blood vessels, nerves, skin, ligaments, tendons, bones, cartilage, and dentin of teeth in vertebrates. , is the main component of the extracellular matrix of multicellular animals. In the present invention, "collagen" mainly refers to type I collagen, and in this specification, a collagen-rich organic composition may be simply referred to as "collagen". In addition, the "collagen" of the present invention is collagen from teeth or bones, and since teeth and bones are tissues that are subject to strong loads such as body weight and occlusal force, collagen from teeth or bones has no ability to support tissues. The highest possible strength is required.

Hard tissue collagen can be determined to be derived from hard tissue by detecting, for example, BMP-2, BMP-4, BMP-7, and osteocalcin; By detecting Dentin Glycoprotein (DGP) and Dentin Phosphoprotein (DPP), it can be determined that the tooth is derived from teeth.

Generally, "collagen-rich organic composition" refers to an organic composition that contains a high proportion of collagen in the entire organic composition, and atelocollagen, demineralized freeze-dried bone (DFDBA), and demineralized dentin matrix (DDM) are , which corresponds to what is generally referred to as a "high collagen content organic composition." In the present invention, the "collagen-rich organic composition" refers to an organic composition in which the proportion of collagen in the whole organic composition is at least 60% or more, and is 70% or more. It is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more. Further, in the present invention, organic compositions other than collagen included in the collagen-rich organic composition include the above-mentioned DSP, DGP, DPP, and the like.

In general, "biomaterials" refer to materials used within the body or materials used in contact with biological components such as proteins and cells. "Biomaterials whose main component is collagen" It refers to a material in which collagen is the main component in the entire material, and decalcified freeze-dried bone (DFDBA) and demineralized dentin matrix (DDM) generally fall under the so-called "biomaterials whose main component is collagen." In the present invention, a "biomaterial containing collagen as a main component" refers to a biomaterial in which the ratio of collagen to the total biomaterial is at least 40%, among biomaterials whose main component is generally collagen. % or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, and most preferably 65% or more. Moreover, examples of organic compositions other than collagen contained in the biomaterial include the above-mentioned DSP, DGP, and DPP.

In the present invention, the pyridinoline and pentosidine contained in the collagen contained in the collagen-rich organic composition are respectively pyridinoline constituting the pyridinoline crosslinked molecule, which is a physiological crosslinked molecule in collagen, and non-physiological crosslinked molecule in collagen. This is the pentosidine that constitutes the pentosidine cross-linked molecule. In the present invention, the amount of pyridinoline contained in collagen contained in the organic composition with high collagen content (ng/mg) and the amount of pentosidine contained in collagen contained in the organic composition with high collagen content (ng/mg) are determined respectively. In addition, by dividing PYD, which is the quantitative value of the amount of pyridinoline contained (ng/mg), by PEN, which is the quantitative value of the amount of pentosidine contained (ng/mg), the amount of pyridinoline contained (ng/mg) is calculated. The weight ratio (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) between PYD, which is the quantitative value of PYD, and PEN, which is the quantitative value of the amount of pentosidine contained (ng/mg), is calculated.

When PYD or PYD and PpP have the above-mentioned predetermined values, the collagen-rich organic composition according to the present invention can be said to be of good quality because the collagen it contains has excellent elasticity and strength. The reason for this will be explained in detail below.

It is no exaggeration to say that pyridinoline crosslinks, which are physiological and enzymatic crosslinking molecules, were programmed primarily to improve the fundamental strength of hard tissues. , is a crosslinking molecule formed between two lysine residues in the telopeptide located at the N-terminus and C-terminus of the amino acid sequence of collagen, and one lysine residue in the amino acid sequence excluding both ends of collagen. . The pyridinoline cross-linked molecules formed between two lysine residues in the telopeptide located at the N-terminus and C-terminus of the amino acid sequence of collagen are cross-linked molecules between collagen fibers, so they have a triple helical structure. It can be said that it greatly contributes to maintaining the three-dimensional structure of pocollagen (the entire collagen arrangement).

Therefore, when the number of pyridinoline cross-linked molecules increases in the collagen contained in a living tissue, the quality of the collagen contained in that living tissue becomes supple and tenacious, the strength and degree of mineralization of the collagen increases, and the composition of the living tissue In other words, increasing the activity of tissue stem cells involved in tissue repair can become an element that helps maintain homeostasis in an individual. Therefore, the amount of pyridinoline contained is higher in collagen derived from teeth and bones than in soft tissue collagen such as skin, and collagen derived from teeth and bones is flexible, has high strength, and has high elasticity.

Collagen contained in the collagen-rich organic composition derived from such pyridinoline cross-linked molecules, that is, collagen-containing pyridinoline contained in teeth or bones extracted from mammals, which are raw materials for the collagen-rich organic composition. The higher the value of PYD (ng/mg), the higher the quality of the collagen contained in the teeth or bones extracted from mammals. Alternatively, a collagen-rich organic composition produced from bone can be said to be an excellent organic composition.

As shown in Figure 1, there are 18 cases of humans, which are mammals, 36 cases of cattle, which are mammals and domestic animals (including 19 cases of bovine SRM (Hokkaido Livestock Corporation, Hayaki Plant), and Tokachi young cattle (Japan). Examples of hard tissues from 17 cases of early fattening male Holsteins (nationally registered trademark, 14 months old; Tokachi Shimizu Town Agricultural Cooperative Association) and 8 cases of domestic pigs (6 months old; Hayaki Plant, Hokkaido Livestock Corporation Co., Ltd.) The PYD, which is the quantitative value of the amount of pyridinoline (ng/mg) contained in the collagen contained in the dentin-derived collagen-rich organic composition of teeth (total of 62 cases), is within the range of PYD according to the present invention. include. Here, "SRM" refers to Specified Risk Materials, and in Japan, it is used to treat the tonsils and distal ileum (part of the small intestine) of all ages, and the head of people over 30 months of age. (excluding tongue and cheek meat), spinal column and spinal cord are designated as specified hazardous areas (Cabinet Office Food Safety Commission).

On the other hand, as shown in Figure 2, soft tissue-derived Telplug (n = 3, atherized collagen derived from bovine dermis, telopeptide-free treatment; Olympus Terumo Biomaterials Co., Ltd.), high-grade gelatin (n = 3, pig Skin acid treatment, telopeptide preserving treatment method, low endotoxin collagen; Nippi Co., Ltd.), Tokachi young cattle (registered trademark in Japan) gingiva (n=3; Tokachi Shimizu Town Agricultural Cooperative), and Tokachi young cattle (Registered Trademark in Japan) muscle (n=3; Tokachi Shimizu Town Agricultural Cooperative Association) has a PYD value of 50 or less, which is a quantitative value of the amount of pyridinoline contained (ng/mg).

From the above, the PYD value according to the present invention is based on the collagen levels derived from tooth dentin, which is the hard tissue, of 18 cases of human humans who are mammals, 36 cases of cows and 8 cases of domestic pigs that are both mammals and domestic animals. From the lower limit of PYD of collagen contained in the containing organic composition, PYD>100 can be achieved.

In other words, in the present invention, the collagen contained in the collagen-rich organic composition, that is, the amount of pyridinoline contained in the collagen contained in the teeth or bones extracted from mammals, which are the raw materials for the collagen-rich organic composition (ng The quantitative value PYD of PYD>100 means that the collagen contained in the collagen-rich organic composition is not collagen derived from mammalian living tissues other than teeth or bones. This means that it can be estimated.

On the other hand, cross-linking by advanced glycation end products (AGEs), typified by pentosidine cross-linking molecules, which are non-physiological cross-linking molecules, is a pathological cross-linking caused by blood sugar (aging cross-linking (AGEs cross-linking)). It is a crosslinking molecule that is formed between arginine and lysine, which are scattered in the amino acid sequence of collagen, when heated by body temperature. When arginine and lysine in the amino acid sequence of collagen are close to each other, pentosidine crosslink molecules are formed nonspecifically (randomly), which can lead to tangled collagen fibers and disrupt the regularity of the collagen fiber arrangement. The increase in pentosidine cross-linked molecules in the amino acid sequence of collagen leads to a decrease in the flexibility, strength, and elasticity of collagen fibers. Since the formation of this pathological cross-linked molecule is caused by blood sugar, it is said that its accumulated amount increases depending on the dietary habits (carbohydrate intake habits) of the host. In addition, since carbohydrates, which are the raw materials for pentosidine cross-linked molecules, are supplied via blood vessels, collagen, which is the support base of tissues throughout the body, is cross-linked evenly and uniformly regardless of the location of the tissue. In other words, it can be said that excessive intake of sugar leads to a uniform decline in the quality of collagen, which serves as the support base of the body, regardless of whether it is in hard or soft tissues.

In addition, when pentosidine cross-linked molecules increase in collagen, the collagen contained in the biological tissue becomes brittle and chalk-like because the pentosidine cross-linked molecules are glycated cross-linked molecules by Maillard reaction, and the collagen contained in the biological tissue becomes weak. and the degree of calcification decreases. Late-stage products of the Maillard reaction are called advanced glycation end products (AGEs), and pentosidine cross-linked molecules are AGEs, and blood sugar levels are maintained at a constant high level due to a diet with high carbohydrate intake. Collagen from mammals with a balanced diet contains more pentosidine cross-linked molecules than collagen from mammals of the same age (months) with a well-balanced diet, which causes blood sugar levels to remain constant. It can be said that aging cross-linking is progressing in mammalian collagen with high levels of collagen. The generated pentosidine cross-linked molecules disrupt the regular arrangement of collagen fibers, impairing the flexibility of collagen, making it hard and brittle, and inhibiting collagen metabolism.As a result, the collagen that makes up the whole body The quality of tissue deteriorates, causing a decrease in tissue suppleness. Collagen is a major component of supporting tissues that support not only all hard tissues such as bones and teeth, but also all soft tissues, including nerves and blood vessels. This can be a factor that deteriorates the quality of soft tissue. For example, when bone quality deteriorates due to bone glycation, the bones become brittle, leading to osteoporosis.In addition, when blood vessel quality decreases due to glycation of blood vessel walls, arteries become hard, so-called arteriosclerosis, which increases blood pressure. It causes tissue damage such as the occurrence of. The three major complications of diabetes are ``(1) diabetic retinopathy (blindness if worsened)'', ``(2) diabetic nephropathy (transition to artificial dialysis if worsened)'', and (3) diabetic neuropathy (deterioration). The same is true for necrosis of fingers and toes due to blood circulation disorder, for example, and the causes are (1) blood circulation disorders in the retina and (2) capillaries in the kidney's glomeruli, which are blood filtering devices. ``vascular disorder'' and ``(3) blood flow disorder at the end of the extremities due to capillary neuropathy and arteriosclerosis,'' both of which can be said to be caused by a decline in quality due to glycation of collagen. be.

In addition, as advanced glycation end products (AGEs), typified by pentosidine cross-linked molecules, increase, AGEs become more likely to bind to RAGE, a receptor for AGEs possessed by cells that make up tissues throughout the body, and as a result, An in-vivo environment is established in which the signal is likely to be activated. This promotes inflammation-related NF-κB signals, and causes chronic inflammation-inducing AGEs, typified by pentosidine cross-linked molecules, to function as chronic inflammation triggering factors in excessively glycated tissues. This mechanism is thought to be a factor that exacerbates chronic inflammatory diseases such as inflammatory bowel disease (IBD) and collagen disease. As described above, collagen saccharification is a factor that gradually erodes living organisms from various directions, unknowingly lowers the quality of life (QOL), and becomes a major factor that prevents living organisms from living long, healthy lives. You get it. It has been reported that both pyridinoline cross-linked molecules and pentosidine cross-linked molecules in collagen contained in human biological tissues increase with aging (Shimizu, ``Non-enzymatic analysis of dentin collagen protein associated with aging'' "Osaka University Knowledge Archive. 2015; Walters C. et al., Calcif. Tissue. Int. Vol. 35, 401-405, 1983).

Collagen contained in the collagen-rich organic composition derived from such pentosidine cross-linked molecules, that is, collagen-containing pentosidine contained in teeth or bones extracted from mammals, which are raw materials for the collagen-rich organic composition. The smaller the value of PEN (ng/mg), the higher the quality of the collagen contained in the teeth or bones extracted from mammals. Alternatively, a collagen-rich organic composition produced from bone can be said to be an excellent organic composition.

On the other hand, humans have a strong tendency to eat miscellaneous foods as they grow, so they tend to accumulate pentosidine cross-linked molecules at a higher rate than healthy livestock animals whose food is maintained at a constant quality throughout their lives. As a result, it is clear that the quality of collagen tends to be inferior, and this tendency is also seen in Figure 3. Therefore, as a standard indicator for the amount of pentosidine contained (ng/mg), , humans should not be used as indicators, but domestic animals that are healthy and live with standard sugar intake habits should be used as indicators.

Therefore, as shown in Figure 3, 44 cases of domestic animals (including 19 cases of bovine SRM (Hokkaido Livestock Corporation Hayaki Plant), 17 cases of Tokachi young cattle (registered trademark in Japan; Tokachi Shimizu Town Agricultural Cooperative Association), The pentosidine content (ng/mg) of the collagen contained in the collagen-rich organic composition derived from the dentin of teeth, which is an example of the hard tissue, of domestic pigs (Hokkaido Livestock Corporation, Hayaki Plant, 8 cases). PEN, which is a quantitative value, can be set to 0<PEN<0.4 from the upper limit values of 44 livestock animals.

In the present invention, the term "domestic animal" refers to a mammal whose feed is managed to have a constant quality, and suitable livestock animals include cows, pigs, horses, sheep, goats, and deer.

In the present invention, the collagen contained in the collagen-rich organic composition derived from pyridinoline cross-linked molecules, that is, the pyridinoline-containing pyridinoline contained in teeth or bones extracted from mammals, which is the raw material for the collagen-rich organic composition. The quantitative value of PYD (ng/mg) and the collagen contained in the collagen-rich organic composition derived from pentosidine crosslinked molecules, that is, the collagen extracted from mammals, which is the raw material of the collagen-rich organic composition. PpP (containing amount of pyridinoline (ng/mg)/containing amount of pentosidine (ng/mg)), which is the value of the weight ratio with PEN, which is the quantitative value of the amount of pentosidine contained in the tooth or bone (ng/mg), is a predetermined value. This value means that the collagen contained in the collagen-rich organic composition or collagen-based biomaterial is flexible, has high strength, and has high elasticity, due to the properties of the pyridinoline crosslinks and pentosidine crosslinks described above. At the same time, it can be estimated that the collagen is of high quality, with the regularity of arrangement and three-dimensional structure of the collagen fibers maintained. However, in this case, it is assumed that PYD is a predetermined value, that is, that the collagen contained in the collagen-rich organic composition or collagen-based biomaterial is not collagen derived from living tissues other than teeth or bones. Become.

In the present invention, the amount of pyridinoline contained in the collagen contained in the collagen-rich organic composition, that is, the collagen contained in the teeth or bones extracted from mammals, which are the raw materials for the collagen-rich organic composition (ng/mg). and the amount of pentosidine contained (ng/mg) can be quantified using a high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. The collagen-rich organic composition according to the present invention can be analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pairing reagents for the collagen contained in the collagen-rich organic composition. PpP (containing pyridinoline amount (ng/mg) )/Amount of pentosidine contained (ng/mg)) can be set to PpP>740 from the lower limit of 44 livestock animals from FIG. 4, since livestock animals are used as an index for PEN.

The samples to be subjected to the determination of the amount of pyridinoline (ng/mg) and pentosidine (ng/mg) contained can be prepared using conventional preparation means known in the art, for example, generally as follows. It can be done as follows.

(1) Dry collagen powder produced by the method for producing a collagen-rich organic composition according to the present invention is weighed into a test tube, and swelled with a small amount of 6N hydrochloric acid under degassing.
(2) After adding 6N hydrochloric acid to the dried collagen and deaerating it, it is sealed.
(3) Hydrolysis is carried out by heat treatment at 110° C. for one day and night.
(4) Cool the sample to room temperature.
(5) Excess hydrochloric acid is removed by drying under reduced pressure while heating to obtain a collagen hydrolyzate.
(6) The obtained collagen hydrolyzate is solubilized with a 10% methanol aqueous solution.
(7) Centrifuge at 12,000 rpm for 5 minutes at room temperature, and use the supernatant as a sample.
(8) By adding a known amount of standard substance to the sample, the extraction rate and calibration curve can be determined, and the collagen content can be corrected as appropriate.

The amount of pyridinoline contained (ng/mg) and the amount of pentosidine contained (ng/mg) were also quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. This can be done using conventional quantitative means known in the art, for example, generally as follows.

(1) Gradient setting using ultrapure water containing 0.05% heptafluorobutyric acid (mobile phase A) and a 1:1 methanol/ethanol mixture containing 0.05% formic acid (mobile phase B) as the mobile phase. Detection is performed by pumping the liquid.
(2) Specific liquid feeding conditions include a detection column temperature of 40° C., a flow rate of 0.5 mL/min, starting with a ratio of mobile phase A of 90% and mobile phase B of 10%, and liquid feeding for 0.5 minutes.
(3) After that, the mobile phase B was increased to 22% over 1 minute to apply a concentration gradient, and then the liquid was fed for 7.5 minutes.
(4) Subsequently, after increasing the mobile phase B to 80% over a period of 1 minute, the liquid was pumped for another 1 minute.
(5) Finally, after feeding at a ratio of mobile phase A of 10% and mobile phase B of 90%, equilibration was performed for 4 minutes at a ratio of mobile phase A of 90% and mobile phase B of 10%.
(6) Perform steps (1) to (5) above for a total of 15 minutes per sample. The separation column used was Cadenza CD-C18, length 150 mm, inner diameter 3 mm, and a fluorescence detector was used for detection. The first 8 minutes are monitored at an excitation wavelength of 295 nm and a fluorescence wavelength of 395 nm, and the latter 7 minutes are monitored at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm. These monitors detect the contained pyridinoline at a retention time of approximately 6.5 minutes, and the contained pentosidine at a retention time of approximately 9.1 minutes.

Next, the collagen-rich organic composition according to the present invention is subjected to a strong alkaline treatment, which is not normally applied to collagen-containing proteins, at a temperature higher than room temperature, and is subjected to a decalcification treatment, that is, an acid treatment. As shown in Figure 6, the amount of endotoxin contained is considerably reduced, or the activity of the contained endotoxin is considerably reduced, resulting in a product that is substantially free of endotoxin or contains no endotoxin. The endotoxin contained therein is not present, or the endotoxin it contains is substantially inactivated, or the endotoxin is inactivated.

A "reduced" amount or activity is typically a "statistically significant" amount or activity that is produced in the absence of the composition (in the absence of drug or compound) or by a control composition. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, including all integers therebetween, in the activity excited by the control composition. 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

"Substantially free of endotoxin" means containing at most trace amounts of endotoxin (e.g., an amount that has no clinically harmful physiological effects on a subject), preferably an undetectable amount of endotoxin. Generally refers to including. Endotoxin is a toxin associated with certain bacteria, typically Gram-negative bacteria, although endotoxin can also be found in Gram-positive bacteria such as Listeria monocytogenes. The most major endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are found in the outer membrane of various Gram-negative bacteria and exhibit central pathogenic characteristics in the ability of these bacteria to cause disease. . Small amounts of endotoxin in humans can cause fever, decreased blood pressure, and activation of inflammation and blood clotting, among other deleterious physiological effects. Therefore, it is often desirable to remove most or all traces of endotoxin from the collagen-rich organic composition of the present invention and the collagen-based biomaterial of the present invention; even can cause harmful effects in humans.

Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus Amebocyte Lysate assay (LAL method), which utilizes blood from horseshoe crabs, is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of lipopolysaccharide (LPS) can cause detectable coagulation of Limulus lysates due to a strong enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). Substantially free of endotoxin, endotoxin levels are approximately 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 , 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0 .022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034 , 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0 .047, 0.048, 0.049, 0.05, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059 , 0.06, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.07, 0.071, 0 .072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.08, 0.081, 0.082, 0.083, 0.084 , 0.085, 0.086, 0.087, 0.088, 0.089, 0.09, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096, 0 .097, 0.098, 0.099, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 , 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0 .32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44 , 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0 .57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 , 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0 .82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94 , 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 , 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3 .1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4 .4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5 .7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 , 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3 , 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6 , 9.7, 9.8, 9.9 or 10 EU/mg or less of protein.

On the other hand, "substantially inactivated" means that the toxicity of the endotoxin is reduced as measured by any standard assay, with the endotoxin being at least 95% inactivated, preferably 98% This means that it is possible to determine that endotoxin is inactivated by 100%, and more preferably by 100%.

Note that biomaterials, biomaterials or other products that use the collagen-rich organic composition according to the present invention as a raw material, collagen-based biomaterials, or other products include biomaterials that use the collagen-rich organic composition according to the present invention as a raw material, There is no particular limitation as long as it is a biomaterial or other product whose main component is collagen.

Note that in order to directly identify the collagen-rich organic composition according to the present invention by its structure and/or properties, a large number of collagen-rich organic compositions according to the present invention are prepared or produced, and various methods are used. However, such tests require a large number of tests and require a large amount of time even for a single competent person skilled in the art. (Patent/Utility Model Examination Handbook, Part II, Chapter 2, Requirements for Description of Claims ``2205.'' A claim for an invention of a product must include a process for manufacturing that product.) (See the bottom line of page 16 to lines 1 to 4 of page 17 of ``Judgment regarding ``impossible/impractical circumstances'' in examinations where In other words, in order to directly identify the collagen-rich organic composition according to the present invention by its structure or properties, it would require unprofitable time and expense for those skilled in the art at the time of filing. Requiring specific work is difficult for applicants who are pursuing the earliest possible filing date under the first-to-file system, in a world where technology is rapidly advancing and competition is intense on an international scale to obtain patents. (Supreme Court No. 1204 of 2012, page 10, or pages 10-11 of Supreme Court No. 2658 of 2012).

Next, a method for producing the collagen-rich organic composition according to the present invention will be explained. Note that among the methods for producing the collagen-rich organic composition according to the present invention, the collagen-rich organic composition produced from teeth or bones extracted from a mammal according to the present invention, as well as the collagen-rich organic composition according to the present invention, are Re-explanation will be omitted for structures that are the same as or correspond to the structures of biomaterials made from organic compositions, biomaterials whose main component is collagen, and other products.

The method for producing the collagen-rich organic composition according to the present invention includes:
(i) A step of crushing a tooth or bone extracted from a mammal to obtain a crushed product (crushed product preparation step);
(ii) a step of treating the pulverized material obtained in the pulverized material preparation step (i) with a strong alkali (strong alkali treatment step);
(iii) a step of deashing the strong alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions (negative pressure deashing step);
It includes the steps (i) to (iii) above.

In the method for producing a collagen-rich organic composition according to the present invention, the pulverized material preparation step (i) involves grinding teeth or bones extracted from a mammal using a conventional pulverizing means known in the art, such as a pulverizer. This is the process of obtaining a pulverized product using In the present invention, the particle size of the pulverized product is preferably 0.05 to 3 mm, more preferably 0.1 to 2 mm.

In the method for producing a collagen-rich organic composition according to the present invention, the strong alkali treatment step (ii) is a stirring treatment of the pulverized material obtained in the pulverized material preparation step (i) in a strongly basic aqueous solution. This is the process of As the strong basic aqueous solution, conventional strong basic aqueous solutions known in the art can be used as appropriate. Examples of such strong basic aqueous solutions include sodium hydroxide aqueous solution and potassium hydroxide. Examples include aqueous solutions, calcium hydroxide aqueous solutions, and sodium carbonate aqueous solutions.

Further, the strong alkali treatment step (ii) is a step of treating the pulverized material obtained in the pulverized material preparation step (i) with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water. Good too. Specifically, the strong alkali treatment step (ii) in the present invention is preferably a step of strong alkali treatment under conditions of 50 to 90°C, and is preferably a step of strong alkali treatment under conditions of 60 to 80°C. It is more preferable to carry out a strong alkali treatment at 65 to 75°C, and most preferably to carry out a strong alkali treatment at 70°C.

In the method for producing a collagen-rich organic composition according to the present invention, the demineralization step (iii) under negative pressure is performed under negative pressure by pulverizing the strongly alkali-treated pulverized material obtained in the strong alkali treatment step (ii). Specifically, it is a process that includes a step of deashing while applying reduced pressure. By performing the deashing process under reduced pressure, the gases generated during the process (mainly CO ₂ ) and the air trapped inside can be removed, so the deashing liquid quickly penetrates into the pulverized material. However, as a result, the time required for demineralization can be shortened. This chemically significant effect allows demineralization to be completed quickly without raising the temperature, thereby indirectly protecting substances that are relatively sensitive to acids. The deashing step (iii) under negative pressure in the present invention may include a step of deashing under reduced pressure, for example, a step of deashing under normal pressure and a step of deashing under reduced pressure. It is also possible to combine the steps of Further, the deashing treatment can be carried out through one to several steps including a conventional deashing step known in the art. For example, the deashing liquid used may be phosphoric acid, hydrochloric acid, nitric acid, , sulfuric acid, formic acid, ethylenediaminetetraacetic acid (EDTA), ethanol and hydrochloric acid, ethanol and EDTA aqueous solution, etc. can be used alone or in combination.

Note that the method for producing the collagen-rich organic composition according to the present invention includes not only the above-mentioned pulverized product preparation step (i), strong alkali treatment step (ii), and negative pressure demineralization step (iii), but also the method of the present invention. Other steps may be included as long as the characteristics of the method are not impaired. Such steps include, for example, a further pulverization step, a drying step, a vacuum drying step, a washing step, a heating step, a cooling step, a neutralization step, a mixing step, an elution step, and the like.

Next, a first embodiment of a method for identifying teeth or bones extracted from a mammal as a raw material for biomaterials, collagen-based biomaterials, or other products according to the present invention will be described. In addition, among the methods of the first embodiment, a collagen-rich organic composition manufactured from teeth or bones extracted from a mammal according to the present invention described above, and a biomaterial using the collagen-rich organic composition as a raw material , biomaterials and other products whose main component is collagen, and structures that are the same as or correspond to the structure of the method for producing the collagen-rich organic composition according to the present invention are given the same reference numerals, etc. Repeated explanation will be omitted.

The method of the first embodiment is as follows:
(iv) a step of quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in teeth or bones extracted from a mammal (step of quantifying the amount of pyridinoline contained);
(v) a step of quantifying the amount of pentosidine (ng/mg) contained in collagen contained in the tooth or bone extracted from the mammal (step of quantifying the amount of pentosidine contained);
(vi) PYD, which is the value of the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained, and the value of the amount of pentosidine contained (ng/mg) determined in the step (v) for determining the amount of pentosidine contained. A step of calculating a weight ratio value of PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) from PEN, which is (weight ratio calculation step),
(vii) PYD, which is the value of the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained, or the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained. When the value PYD and the weight ratio value PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg)) calculated in the weight ratio calculation step (vi) are predetermined values. a step of selecting the tooth or bone extracted from the mammal as a biomaterial, a biomaterial containing collagen as a main component, or a raw material for other products (teeth/bone selection step);
It includes the steps (iv) to (vii) above.

In the step (iv) of quantifying the amount of pyridinoline contained and the step (v) of quantifying the amount of pentosidine contained in the method of the first embodiment, the amount of pyridinoline contained (ng/mg) in collagen contained in teeth or bones extracted from a mammal is determined. The method for quantifying the amount of pentosidine contained (ng/mg) is not particularly limited as long as it is possible to quantify it, but examples of such a method include, for example, using heptafluorobutyric acid and formic acid as ion pairing reagents. A high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) can be employed.

In the weight ratio calculation step (vi) in the method of the first embodiment, the amount of pyridinoline contained in the collagen contained in the tooth or bone extracted from the mammal (ng/mg) is determined in the step (iv) of determining the amount of pyridinoline contained. ) is the quantitative value of PYD, which is the quantitative value of pentosidine content (ng/mg), and PEN, which is the quantitative value of the pentosidine content (ng/mg) of collagen contained in the teeth or bones extracted from mammals, which was quantified in the pentosidine content determination step (v). By dividing, PpP (containing pyridinoline amount (ng/mg)/containing pentosidine amount (ng/mg)).

In the tooth/bone selection step (vii) in the method of the first embodiment, PYD, which is the value of the amount of pyridinoline contained (ng/mg) determined in the step (iv) of quantifying the amount of pyridinoline contained, or the step of quantifying the amount of pyridinoline contained PYD, which is the value of the amount of pyridinoline contained (ng/mg) quantified in (iv), and PpP, which is the value of the weight ratio calculated in step (vi) of calculating the weight ratio (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained) (ng/mg)) is a predetermined value, the tooth or bone extracted from the mammal is selected as a biomaterial, a collagen-based biomaterial, or a raw material for other products. .

The "predetermined value" referred to in the tooth/bone selection step (vii) refers to the amount of pyridinoline (ng/mg) and the amount of pentosidine (ng/mg) contained in collagen contained in teeth or bones extracted from mammals. For example, the step (iv) of quantifying the amount of pyridinoline contained and the step (v) of quantifying the amount of pentosidine contained are performed using a high performance liquid chromatograph with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pairing reagents. (HPLC-Flu), the predetermined values are PYD>100 or PYD>100 and PpP>740, respectively.

Note that the method of the first embodiment is similar to the method of manufacturing the collagen-rich organic composition according to the present invention, including the above-described step (iv) of determining the amount of pyridinoline contained, the step (v) of determining the amount of pentosidine contained, and the weight In addition to the ratio calculation step (vi) and the tooth/bone selection step (vii), other steps may be included as long as the characteristics of the present invention are not impaired.

Next, a second embodiment of a method for identifying teeth or bones extracted from a mammal as raw materials for biomaterials, collagen-based biomaterials, or other products according to the present invention will be described. The method of the second embodiment is as follows:
(i) A step of crushing a tooth or bone extracted from a mammal to obtain a crushed product (crushed product preparation step);
(ii) a step of treating the pulverized material obtained in the pulverized material preparation step (i) with a strong alkali (strong alkali treatment step);
(iii) a step of decalcifying the strongly alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions to obtain a collagen-rich organic composition (demineralization step under negative pressure);
(iv) a step of quantifying the amount of pyridinoline (ng/mg) contained in the collagen contained in the collagen-rich organic composition obtained in the negative pressure demineralization step (iii) (step of quantifying the amount of pyridinoline contained);
(v) a step of quantifying the amount of pentosidine (ng/mg) contained in collagen contained in the collagen-rich organic composition obtained in the negative pressure demineralization step (iii) (step of quantifying the amount of pentosidine contained);
(vi) PYD, which is the value of the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained, and the value of the amount of pentosidine contained (ng/mg) determined in the step (v) for determining the amount of pentosidine contained. A step of calculating a weight ratio value of PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) from PEN, which is (weight ratio calculation step),
(vii) PYD, which is the value of the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained, or the amount of pyridinoline contained (ng/mg) determined in the step (iv) for determining the amount of pyridinoline contained. When the value PYD and the weight ratio value PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg)) calculated in the weight ratio calculation step (vi) are predetermined values. a step of selecting teeth or bones extracted from the mammal as raw materials for biomaterials, collagen-based biomaterials, or other products (teeth/bone selection step);
It has the steps (i) to (vii) above. In addition, among the methods of the second embodiment, a collagen-rich organic composition manufactured from teeth or bones extracted from a mammal according to the present invention described above, and a biomaterial using the collagen-rich organic composition as a raw material , biomaterials and other products containing collagen as a main component, methods for producing the collagen-rich organic composition according to the present invention, and biomaterials, biomaterials containing collagen as a main component, and other products as raw materials. Components that are the same as or correspond to the configurations of the first embodiment of the method for identifying teeth or bones extracted from mammals are given the same reference numerals and will not be described again.

The pulverized product preparation step (i), strong alkali treatment step (ii), and negative pressure demineralization step (iii), which are the constituent elements of the method of the second embodiment, are performed using the collagen-rich organic composition according to the present invention. This corresponds to the pulverized product preparation step (i), strong alkali treatment step (ii), and negative pressure demineralization step (iii), which are the constituent elements of the manufacturing method, and is also a constituent element of the method of the second embodiment. The steps of determining the amount of pyridinoline contained (iv), the step of determining the amount of pentosidine contained (v), the weight ratio calculation step (vi), and the tooth/bone selection step (vii) are based on the biomaterial according to the present invention, which mainly contains collagen. Step (iv) of quantifying the amount of pyridinoline contained, step (iv) of quantifying the amount of pentosidine contained, which are the constituent elements of the first embodiment of the method for identifying teeth or bones extracted from mammals as raw materials for biomaterials or other products. v), weight ratio calculation step (vi), and tooth/bone selection step (vii).

Note that the method of the second embodiment also includes a method for producing a collagen-rich organic composition according to the present invention, and a raw material for biomaterials, collagen-based biomaterials, or other products according to the present invention. Similarly to the first embodiment of the method for identifying teeth or bones extracted from a mammal, the above-mentioned pulverized product preparation step (i), strong alkali treatment step (ii), and negative pressure demineralization step (iii) ), the step of quantifying the amount of pyridinoline contained (iv), the step of quantifying the amount of pentosidine contained (v), the weight ratio calculation step (vi), and the tooth/bone selection step (vii), as well as within the range that does not impair the characteristics of the present invention. It may also include other steps.

Next, as an index for evaluating the potential risk of disease in a mammal according to the present invention and/or whether or not the mammal is potentially healthy, pyridinoline contained in collagen contained in the teeth of a mammal is used. Calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the quantitative value of the amount of pentosidine contained in collagen contained in mammalian teeth. The method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained) is
(viii) Quantifying the amount (ng/mg) of pyridinoline contained in collagen contained in the teeth of a mammal, which is the target of evaluating the potential risk of disease and/or whether or not the mammal is potentially healthy. and obtaining a quantitative value of PYD (quantification process of the amount of pyridinoline contained in the evaluation target),
(ix) a step of quantifying the amount of pentosidine (ng/mg) contained in the collagen contained in the teeth of the mammal to be evaluated to obtain a quantitative value of PEN (quantification step of the amount of pentosidine contained in the evaluation target);
(x) From the PYD obtained in the step (viii) for quantifying the amount of pyridinoline contained in the evaluation target and the PEN obtained in the step (ix) for quantifying the amount of pentosidine contained in the evaluation target, the weight ratio value of PpP (the amount of pyridinoline contained ( ng/mg)/contained pentosidine amount (ng/mg)) (evaluation target weight ratio calculation step),
(xi) collagen contained in the teeth of one or more mammals selected from mammals not suffering from diseases, mammals suffering from diseases, healthy mammals, and unhealthy mammals; A step of quantifying the amount of pyridinoline contained (ng/mg) to obtain R-PYD, which is a reference quantitative value (a step of quantifying the amount of reference pyridinoline contained),
(xii) collagen contained in the teeth of one or more mammals selected from mammals not suffering from diseases, mammals suffering from diseases, healthy mammals, and unhealthy mammals; A step of quantifying the amount of pentosidine contained (ng/mg) to obtain R-PEN, which is a reference quantitative value (reference step of quantifying the amount of pentosidine contained),
(xiii) From the R-PYD obtained in the reference-containing pyridinoline amount determination step (xi) and the R-PEN obtained in the reference-containing pentosidine amount determination step (xii), a weight ratio reference value of R-PpP is determined. Step of calculating (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) (reference weight ratio calculation step),
(xiv) Comparing the PYD obtained in the evaluation target content pyridinoline amount determination step (viii) and the R-PYD obtained in the reference content pyridinoline amount determination step (xi), or the evaluation target content pyridinoline amount determination step PYD obtained in (viii) and R-PYD obtained in the reference-containing pyridinoline amount determination step (xi), PpP calculated in the evaluation target weight ratio calculation step (x) and reference weight ratio calculation step (xiii) ) A step of comparing R-PpP calculated in (PYD/weight ratio comparison step),
The above steps (viii) to (xiv) are included.

In addition, as an index for evaluating the potential risk of disease in a mammal according to the present invention and/or whether or not the mammal is potentially healthy, the amount of pyridinoline contained in collagen contained in the teeth of the mammal is used. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. Among the methods using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained), the collagen-rich organic composition manufactured from teeth or bones extracted from a mammal according to the present invention, the collagen-rich organic composition Biomaterials as raw materials, biomaterials containing collagen as a main component, and other products, methods for producing the collagen-rich organic composition according to the present invention, and biomaterials, biomaterials containing collagen as a main component, and other products. Structures that are the same as or equivalent to those of the first and second embodiments of the method for identifying teeth or bones extracted from mammals as raw materials for products will be described again with the same reference numerals. omitted.

In the present invention, "potential disease risk" does not refer to the state of being affected by a disease, but rather the state in which the host's defense response to the disease has worked to its maximum and the disease has not yet reached its potential. A risky state in which the presence of a disease cannot be confirmed based on subjective, objective, or conventional test results.

In addition, in the present invention, "potentially healthy" refers to a state that does not lead to an unhealthy state, and is almost always unhealthy based on subjective, objective findings, and conventional general test findings. In the present invention, "potentially unhealthy" refers to a state that does not lead to a state of being healthy, and is a state that does not lead to a state of being healthy, even when subjective, objective findings, and conventional general test findings indicate that it is not healthy. It means a state in which it is almost impossible to confirm that

Quantification of the amount of pyridinoline contained in collagen contained in the teeth of a mammal as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the quantitative value of the amount of pentosidine contained in collagen. In the method using (amount of pyridinoline contained/amount of pentosidine contained), the step (viii) of quantifying the amount of pyridinoline to be evaluated is performed as a raw material for biomaterials according to the present invention, biomaterials mainly composed of collagen, or other products. This step corresponds to the step (iv) of quantifying the amount of pyridinoline contained in the first and second embodiments of the method for identifying teeth or bones extracted from a mammal.

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (amount of pyridinoline contained/amount of pentosidine contained), the step (ix) of quantifying the amount of pentosidine to be evaluated is performed as a raw material for the biomaterial according to the present invention, a biomaterial mainly composed of collagen, or other products. This step corresponds to the step (v) of quantifying the amount of pentosidine contained in the first and second embodiments of the method for identifying teeth or bones extracted from a mammal.

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in a mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (contained pyridinoline amount/contained pentosidine amount), the evaluation target weight ratio calculation step (x) is the evaluation target weight ratio calculation step (x) of the biomaterial according to the present invention, the biomaterial mainly composed of collagen, or the raw material of other products. This step corresponds to the weight ratio calculation step (vi) of the first and second embodiments of the method for identifying teeth or bones extracted from a mammal.

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in a mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained), the step (xi) of quantifying the amount of reference pyridinoline contains the amount of pyridinoline used as a raw material for the biomaterial according to the present invention, the biomaterial mainly composed of collagen, or other products. , the step (iv) of quantifying the amount of pyridinoline contained in the first and second embodiments of the method for differentiating teeth or bones extracted from a mammal, and the potential risk of disease in the mammal according to the present invention. / Or as an index for evaluating whether or not the mammal is potentially healthy, quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal, or content of collagen contained in the teeth of the mammal In a method using a quantitative value of the amount of pyridinoline, and a weight ratio (amount of pyridinoline contained/amount of pentosidine contained) calculated from a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and a quantitative value of the amount of pentosidine contained, This corresponds to the step (viii) of quantifying the amount of pyridinoline contained in the evaluation target.

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (amount of pyridinoline contained/amount of pentosidine contained), the step (xii) of quantifying the amount of reference pentosidine contains the biomaterial of the present invention, the biomaterial mainly composed of collagen, or the amount of pentosidine used as a raw material for other products. , the step (v) of quantifying the amount of pentosidine contained in the first and second embodiments of the method for differentiating teeth or bones extracted from a mammal, and the potential risk of disease in the mammal according to the present invention. / Or as an indicator for evaluating whether or not a mammal is potentially healthy, a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, or the content of collagen contained in the teeth of a mammal. In a method using a quantitative value of the amount of pyridinoline and a weight ratio (amount of pyridinoline contained/amount of pentosidine contained) calculated from a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and a quantitative value of the amount of pentosidine contained, This corresponds to the step (ix) of quantifying the amount of pentosidine contained in the evaluation target.

In addition, in the reference-containing pentosidine amount determination step (xii), "one or more selected from mammals not suffering from a disease, mammals suffering from a disease, healthy mammals, and unhealthy mammals" "Collagen contained in mammalian teeth" is different from the step (xi) for quantifying the amount of pyridinoline containing reference. It may be "collagen contained in the teeth of one or more mammals selected from mammals", and the same "mammals not suffering from a disease, mammals suffering from a disease, healthy mammals" The collagen contained in the teeth of one or more mammals selected from animals and unhealthy mammals may also be used.

In addition, in any of the steps, "a mammal not suffering from a disease" includes "self, which is a mammal not suffering from a disease", and "a mammal suffering from a disease" includes "a mammal not suffering from a disease". ``self that is a mammal that is suffering from a disease,'' ``healthy mammal'' includes ``self that is a healthy mammal,'' and ``unhealthy mammal'' includes ``self that is a healthy mammal.'' ``self that is a mammal'' is included.

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained), the reference weight ratio calculation step (xiii) is the biomaterial according to the present invention, the biomaterial mainly composed of collagen, or as a raw material for other products. Weight ratio calculation step (vi) of the first and second embodiments of the method for differentiating teeth or bones extracted from a mammal, and the potential risk of disease and/or disease in the mammal according to the present invention As an indicator for evaluating whether or not a mammal is potentially healthy, quantitative values of the amount of pyridinoline contained in collagen contained in mammalian teeth or the amount of pyridinoline contained in collagen contained in mammalian teeth are used. Evaluation target in a method using the quantitative value of , and the weight ratio (containing pyridinoline amount/containing pentosidine amount) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and the quantitative value of the amount of pentosidine contained. This corresponds to the weight ratio calculation step (x).

In addition, the amount of pyridinoline contained in collagen contained in the teeth of a mammal is used as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. In the method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained), the PYD/weight ratio comparison step (xiv) is based on PpP (the amount of pyridinoline contained), which is the value of the weight ratio calculated in the evaluation target weight ratio calculation step (x). (ng/mg)/Amount of pentosidine contained (ng/mg)) and R-PpP (Amount of pyridinoline contained (ng/mg)/Amount of pentosidine contained), which is the reference value of the weight ratio calculated in the reference weight ratio calculation step (xiii). (ng/mg)).

Quantification of the amount of pyridinoline contained in collagen contained in the teeth of a mammal as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and the quantitative value of the amount of pentosidine contained. In the method using (amount of pyridinoline contained/amount of pentosidine contained), a step (viii) for quantifying the amount of pyridinoline contained in the evaluation target, a step (ix) in quantifying the amount of pentosidine contained in the evaluation target, a step (xi) for quantifying the amount of pyridinoline contained in the reference, and the amount of pentosidine contained in the reference The quantitative step (xii) is carried out in mammals to be evaluated for potential risk of contracting the disease, mammals not suffering from the disease, mammals suffering from the disease, healthy mammals, and non-healthy mammals. In addition to being able to be carried out on extracted teeth of mammals, it can also be carried out in a manner such as cutting, contact, or non-contact.

In addition, as an index for evaluating the potential risk of disease in a mammal according to the present invention and/or whether or not the mammal is potentially healthy, the amount of pyridinoline contained in collagen contained in the teeth of the mammal is used. or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the amount of pentosidine contained in collagen. The method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained) is also applicable to the method for producing the collagen-rich organic composition according to the present invention, as well as the biomaterial according to the present invention, the biomaterial mainly composed of collagen, or Similar to the first and second embodiments of the method for identifying teeth or bones extracted from mammals as raw materials for other products, the step (viii) of quantifying the amount of pyridinoline contained in the evaluation target described above, Contained pentosidine amount determination step (ix), evaluation target weight ratio calculation step (x), reference contained pyridinoline amount determination step (xi), reference contained pentosidine amount determination step (xii), reference weight ratio calculation step (xiii), and PYD・In addition to the weight ratio comparison step (xiv), other steps may be included as long as the features of the present invention are not impaired.

Quantification of the amount of pyridinoline contained in collagen contained in the teeth of a mammal as an index for evaluating the potential risk of disease in the mammal according to the present invention and/or whether or not the mammal is potentially healthy. or a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the quantitative value of the amount of pentosidine contained in collagen. The reason why the method using (amount of pyridinoline/amount of pentosidine contained) is limited to "teeth" is that both pyridinoline crosslinks and pentosidine crosslinks in type I collagen are supplied through internal heat from body temperature and through blood vessels. This is a cross-linking reaction that is accelerated by factors such as sugar, and is not local but uniform throughout the body.Among these, the cross-linking reaction is thought to most accurately reflect the accumulation of pyridinoline cross-linked molecules and pentosidine cross-linked molecules. This is because collagen is contained in teeth (especially dentin). Teeth are the only tissue in the living body that is not incorporated into the metabolic cycle, and once formed, they are not absorbed or degraded. Therefore, pyridinoline cross-linked molecules and pentosidine cross-linked molecules are used in collagen contained in teeth (particularly dentin). This is because it is accumulated in

Additionally, collagen in teeth (especially dentin) is insoluble compared to bone. Pepsin digestion under the conditions of 0.01N hydrochloric acid, 4°C, and 72 hours of treatment time solubilizes approximately 35% of collagen in adult bovine bone, but 5.6% in dentin of adult bovine teeth. Only % of collagen is solubilized. In addition, under conditions of pH 2, insoluble collagen in skin and Achilles tendon swells to 4 to 8 times its volume, and insoluble collagen in adult bovine bone swells to 1.2 times its volume, whereas adult bovine dentin swells to 1.2 times its volume. Insoluble collagen does not swell at all (Yutaka Nagai and Daizaburo Fujimoto, eds., Collagen Experimental Methods, Kodansha Scientific, p. 21-22). Additionally, tooth dentin collagen has the highest content of pyridinoline crosslinked molecules in living organisms. Therefore, PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and Diseases in mammals are determined using PpP (pyridinoline content/pentosidine content), which is a weight ratio value calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen, and PEN, which is a quantitative value of the amount of pentosidine contained in collagen. It makes sense to assess the potential risk of morbidity and/or whether the mammal is potentially healthy.

On the other hand, the basis for excluding bones will be explained. The values of type I collagen C-terminal telopeptide (ICTP) in blood (serum) and NTx (type I collagen cross-linked N-telopeptide) in urine are widely used as indicators of osteoporosis; It is a substance that is released into the blood and urine as a collagen degradation product due to the activity of metalloproteases (MMPs), and it is a substance that contains pyridinoline crosslinked molecules. This indicates that pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained in bone-containing collagen are partially cleared through metabolism. This is because the organ contains a large amount of lipid, which makes it difficult to degrease it and it is extremely difficult to purify highly pure collagen.

In addition, if the amount of pyridinoline contained in the collagen contained in the teeth of a mammal is large and the amount of pentosidine contained is small, the mammal has collagen that improves its own homeostasis and is less likely to cause chronic inflammation. Therefore, it can be evaluated that the risk of contracting various latent diseases is low, and it can also be determined that the person is potentially healthy. On the other hand, if the amount of pyridinoline contained in the collagen contained in the teeth of a mammal is small and the amount of pentosidine contained is large, the mammal has collagen that reduces its own homeostasis and is likely to cause chronic inflammation. The potential risk of contracting a disease in a mammal and/or the mammal can be assessed as having a high risk of contracting a variety of potential diseases, and can be determined to be potentially unhealthy. PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or the pyridinoline content of collagen contained in mammalian teeth, is used as an index for evaluating whether or not the teeth are potentially healthy. PYD, which is a quantitative value of the amount, and PpP, which is a weight ratio value calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PEN, which is a quantitative value of the amount of pentosidine contained. (amount of pyridinoline contained/amount of pentosidine contained) can be used. As an index for evaluating the potential risk of disease in mammals according to the present invention, PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, is used. Weight calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PEN, which is a quantitative value of the amount of pentosidine contained. A method using PpP (amount of pyridinoline contained/amount of pentosidine contained), which is a ratio value, and as an index for evaluating whether or not a mammal according to the present invention is potentially healthy, PYD is a quantitative value of the amount of pyridinoline contained in collagen, or PYD is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PYD is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth. The methods using PYD, which is a quantitative value, and PpP, which is a weight ratio value calculated from PEN, which is a quantitative value of the amount of pentosidine contained, (contained pyridinoline amount / contained pentosidine amount) may be independent. , may be related.

Hereinafter, a collagen-rich organic composition manufactured from a tooth or bone extracted from a mammal, a biomaterial using the collagen-rich organic composition as a raw material, a biomaterial containing collagen as a main component, and others according to the present invention will be described below. Differentiation of teeth or bones extracted from mammals as raw materials for products, methods for producing collagen-rich organic compositions according to the present invention, biomaterials, biomaterials containing collagen as a main component, or other products. The method and the content of collagen in the teeth of a mammal as an indicator for evaluating the potential risk of disease in a mammal and/or whether or not the mammal is potentially healthy according to the present invention Calculated from the quantitative value of the amount of pyridinoline, or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the quantitative value of the quantitative value of the amount of pentosidine contained in collagen. A method using the weight ratio (the amount of pyridinoline contained/the amount of pentosidine contained) will be explained based on Examples. Note that the technical scope of the present invention is not limited to the embodiments shown by these Examples.

<<Example 1>> Measurement of the amount of pyridinoline and pentosidine in collagen derived from human, bovine, and porcine teeth (mainly dentin) The following procedure was performed in 18 human and 36 bovine cases (of which bovine SRM (Co., Ltd.) 19 cases were caused by Hokkaido Livestock Corporation Hayaki Plant), 17 cases were caused by Tokachi young cattle (registered trademark in Japan, early fattening (14 months old) male Holstein; Tokachi Shimizu Town Agricultural Cooperative Association), and domestic pigs (6 months old; (Hokkaido Livestock Corporation Hayaki Plant) 8 cases of collagen derived from teeth (mainly dentin) (n = 62), Telplug derived from soft tissue (n = 3, atherized collagen derived from bovine dermis, telopeptide-free treatment; Olympus Terumo Biomaterial Co., Ltd.), high-grade gelatin (n = 3, porcine acid treatment, telopeptide preservation treatment method, low endotoxin collagen; Nippi Co., Ltd.), Tokachi young beef (registered trademark in Japan) gingiva ( The amounts of pyridinoline and pentosidine were measured in the muscles of Tokachi young cattle (n=3; Tokachi Shimizu Town Agricultural Cooperative Association) and the muscles of Tokachi young cattle (n=3; Tokachi Shimizu Town Agricultural Cooperative Association).

1. Preparation of Collagen Powder [1-1] Hard tissue was ground to a particle size of about 1 mm using a grinder.
[1-2] Adhering soft tissues were removed by stirring in a 1% aqueous sodium carbonate solution at 70°C for several hours.
[1-3] Decalcification treatment was performed. Specifically, after deashing for several hours while reducing the pressure using a deashing solution containing 1M phosphoric acid and ethanol, the deashing solution is replaced with the deashing solution diluted several times, and the pressure is reduced. Demineralization treatment was carried out for several hours. Subsequently, the above-mentioned decalcification solution diluted five times was used to completely demineralize by shaking at room temperature for about a day and night. Thereafter, washing was performed several times using 5% ethanol at room temperature.
[1-4] The hard tissue after decalcification was transferred to a mortar and ground in 70% ethanol to form a fine powder.
[1-5] Washing, degreasing, and dehydration were performed by stirring with 100% ethanol.
[1-6] Ethanol remaining in the collagen was completely removed by drying to obtain dry collagen powder.

2. Preparation of sample for analysis [2-1] The dried collagen powder obtained in [1-6] was hydrolyzed. In detail, the obtained dry collagen powder was weighed into a test tube, swelled sufficiently with a small amount of 6N hydrochloric acid under deaerated conditions, an appropriate amount of 6N hydrochloric acid was added to the dried collagen, and the test tube was placed under deaerated conditions. Welded and sealed. Hydrolyze the sealed test tube by heating it at 110°C overnight, cool it to room temperature, cut the test tube, and dry under reduced pressure while heating to remove the excess. Hydrochloric acid was removed to obtain a collagen hydrolyzate.
[2-2] Solubilize the obtained collagen hydrolyzate with a 10% methanol aqueous solution,
The mixture was centrifuged at 12,000 rpm for 5 minutes at room temperature, and the supernatant was used as a sample.
[2-3] By adding a known amount of standard substance to the sample, the extraction rate and calibration curve were determined, and the collagen content was corrected as appropriate.

3. Quantification of the amount of pyridinoline contained and amount of pentosidine contained and calculation of their weight ratio by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents 0.05% as mobile phase Ultrapure water containing heptafluorobutyric acid (mobile phase A) and a 1:1 mixture of methanol and ethanol containing 0.05% formic acid (mobile phase B) were used to send the liquid in a gradient setting for detection. Specifically, at a detection column temperature of 40° C. and a flow rate of 0.5 mL/min, starting with a ratio of mobile phase A of 90% and mobile phase B of 10%, the liquid was pumped for 0.5 minutes. Thereafter, the mobile phase B was increased to 22% over 1 minute to apply a concentration gradient, and then the solution was fed for 7.5 minutes. Subsequently, the mobile phase B was increased to 80% over 1 minute, and then the liquid was pumped for another 1 minute. Finally, after feeding at a ratio of 10% mobile phase A and 90% mobile phase B, the mobile phase B was subsequently lowered to 10% and equilibrated for 4 minutes at a ratio of 90% mobile phase A and 10% mobile phase B. Subsequently, detection was performed for a total of 15 minutes per sample. A Cadenza CD-C18 with a length of 150 mm and an inner diameter of 3 mm was used as a separation column, and a fluorescence detector was used for detection. The first 8 minutes were monitored at an excitation wavelength of 295 nm and a fluorescence wavelength of 395 nm, and the latter 7 minutes were monitored at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm. The monitor detected the contained pyridinoline at a retention time of approximately 6.5 minutes, and the contained pentosidine at a retention time of approximately 9.1 minutes. From the obtained pyridinoline content and pentosidine content, their weight ratio (pyridinoline content (ng/mg)/pentosidine content (ng/mg)) was determined.

Regarding human teeth, the measurement target is permanent teeth, but if we take into account the period of formation of dentin in permanent teeth, dentin formation starts in the tooth germ in the jawbone from around the age of 10 on average, and along with this, glycation It can be considered that the formation of crosslinks has also started (Survey Study II on the eruption timing of deciduous teeth and permanent teeth in Japanese children, Journal of the Japanese Society of Pediatric Dentistry, 57(3), 363-373, 2019, 363). On the other hand, since the same can be said about the teeth in the lower jaw of bovine SRM after 18 months of age on average, the following formula for converting from age to age in months can be used for certain teeth of human and bovine SRM. Table 1 was used.

[Table 1]

The amount of pyridinoline contained in an organic composition with a high content of collagen derived from the dentin of 18 human teeth, determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. PYD is the quantitative value of (ng/mg), PEN is the quantitative value of the amount of pentosidine contained (ng/mg), and PYD is the quantitative value of the amount of pyridinoline contained (ng/mg) and the amount of pentosidine contained (ng/mg). PpP (containing pyridinoline amount (ng/mg)/containing pentosidine amount (ng/mg)), which is the weight ratio value calculated from PEN, which is the quantitative value of mg), is shown in Table 2 below.

[Table 2]

The amount of pyridinoline contained in an organic composition with a high content of collagen derived from the dentin of 19 cases of bovine SRM, determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. PYD is the quantitative value of (ng/mg), PEN is the quantitative value of the amount of pentosidine contained (ng/mg), and PYD is the quantitative value of the amount of pyridinoline contained (ng/mg) and the amount of pentosidine contained (ng/mg). PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)), which is a value of the weight ratio calculated from PEN, which is a quantitative value of mg), is shown in Table 3 below.

[Table 3]

A high content of organic collagen derived from the dentin of 17 teeth of Tokachi young cattle (registered trademark in Japan), determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. PYD, which is a quantitative value of the amount of pyridinoline contained in the composition (ng/mg), PEN, which is a quantitative value of the amount of pentosidine contained (ng/mg), and a quantitative value of the amount of pyridinoline contained (ng/mg). PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg)), which is the value of the weight ratio calculated from PYD and PEN, which is the quantitative value of the contained pentosidine amount (ng/mg), is shown below. It is shown in Table 4.

[Table 4]

Pyridinoline contained in an organic composition with a high content of collagen derived from the dentin of the teeth of eight domestic pigs, as determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. PYD, which is a quantitative value of the amount of pentosidine contained (ng/mg), PEN, which is a quantitative value of the amount of pentosidine contained (ng/mg), and PYD, which is a quantitative value of the amount of pyridinoline contained (ng/mg), and the amount of pentosidine contained (ng/mg). Table 5 below shows PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)), which is a weight ratio value calculated from PEN, which is a quantitative value of /mg).

[Table 5]

In addition, a total of 62 cases of high collagen content derived from dentin were found in the teeth of 18 cases of humans, 36 cases of cattle (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs. PYD is a quantitative value of the amount of pyridinoline (ng/mg) contained in collagen contained in an organic composition, measured by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. Figure 1 shows the distribution of dentine derived from the teeth of 18 humans, 36 cows (of which 19 were bovine SRM, and 17 were Tokachi young cattle (registered trademark in Japan)), and 8 domestic pigs. Collagen (n=62) contained in the collagen-rich organic composition, Telplug derived from soft tissue (n=3, derived from bovine dermis), high-grade gelatin (n=3), and gingiva of Tokachi young cattle (registered trademark in Japan) (n=3) and collagen contained in Tokachi young cattle (registered trademark in Japan) muscle (n=3) using high performance liquid chromatography with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pairing reagents. The distribution of PYD, which is the quantitative value of the amount of pyridinoline contained (ng/mg) determined by (HPLC-Flu), is shown in FIG.

As shown in Figure 1, there are 18 human cases that are mammals, 36 cases of cows that are both mammals and domestic animals (including 19 cases of bovine SRM, 17 cases of Tokachi young cattle (registered trademark in Japan)), and 8 cases of domestic pigs. PYD, which is a quantitative value of the amount of pyridinoline contained in collagen (ng/mg) contained in the organic composition with high collagen content derived from dentin, which is an example of hard tissue (62 cases in total), is as follows. It was included in the scope of PYD according to the present invention.

On the other hand, as shown in Figure 2, soft tissue-derived telplug (n = 3, derived from bovine dermis), high-grade gelatin (n = 3), Tokachi young cow (registered trademark in Japan) gingiva (n = 3), and The PYD, which is a quantitative value of the amount of pyridinoline contained (ng/mg), in the muscles (n=3) of Tokachi Wakagyu (registered trademark in Japan) were all 50 or less.

From the above, the PYD value according to the present invention is based on the collagen levels derived from tooth dentin, which is the hard tissue, of 18 cases of human humans who are mammals, 36 cases of cows and 8 cases of domestic pigs that are both mammals and domestic animals. Based on the lower limit of PYD of collagen contained in the containing organic composition, PYD>100 was set.

In addition, a total of 62 cases of high collagen content derived from dentin were found in the teeth of 18 cases of humans, 36 cases of cattle (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs. The quantitative value of the amount of pentosidine (ng/mg) contained in the collagen contained in the organic composition, determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. The distribution of a certain PEN is shown in FIG.

As shown in Figure 3, collagen derived from tooth dentin, which is an example of hard tissue, was obtained from 44 domestic animals (including 19 cases of bovine SRM, 17 cases of Tokachi young cattle (registered trademark in Japan), and 8 cases of domestic pigs). The PEN, which is a quantitative value of the pentosidine content (ng/mg) of the collagen contained in the high-content organic composition, was determined to be 0<PEN<0.4 based on the upper limit value of 44 livestock animals.

In addition, a total of 62 cases of high collagen content derived from dentin were found in the teeth of 18 cases of humans, 36 cases of cattle (including 19 cases of bovine SRM and 17 cases of Tokachi young cattle (registered trademark in Japan)) and 8 cases of domestic pigs. The quantitative value of the amount of pyridinoline (ng/mg) contained in the collagen contained in the organic composition, determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. The value of PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg)), which is the value of the weight ratio calculated from a certain PYD and PEN, which is the quantitative value of the contained pentosidine amount (ng/mg) The distribution of is shown in Figure 4.

As shown in FIG. 4, PpP (containing pyridinoline amount ( ng/mg)/Amount of pentosidine contained (ng/mg)) was set as PpP>740 since livestock animals are used as an indicator for PEN.

<<Example 2>> Endotoxin test on collagen derived from bovine teeth (mainly dentin), gelatin derived from pig skin, and high-grade gelatin Tokachi young cattle (registered trademark in Japan, early fattened (14 months old) male Holstein; Tokachi Endotoxin tests were conducted on 6 cases of collagen derived from teeth (mainly dentin) obtained from Shimizu Town Agricultural Cooperative Association, 3 cases of gelatin derived from pig skin (gelatin from porcine skin, Merck), and 3 cases of high-grade gelatin. The endotoxin test was performed colorimetrically using the LAL reagent made from Limulus amebocyte lysate using horseshoe crab-derived blood. The results are shown in FIG.

In addition, samples of tooth-derived collagen from 6 cases of Tokachi Wakagyu (registered trademark in Japan) were prepared by the following method.
(1) For collagen with residual mineral components, use a decalcification solution containing 500mM EDTA and shake at 37°C for 2 days while replacing the decalcification solution to completely remove the mineral components. , EDTA was removed by washing with ultrapure water.
(2) The obtained collagen was completely dried to obtain dried collagen.
(3) The dried collagen was completely degraded using Proteinase K (Takara Bio Inc.) at 56°C.
(4) Proteinase K was then deactivated by heating at 95° C. for 5 minutes.
(5) Centrifugation was performed at 12,000 rpm at room temperature, and the supernatant was used as a collagen sample.

As shown in Figure 5, the endotoxin level of tooth-derived collagen from 6 cases of Tokachi Wakagyu (registered trademark in Japan) was less than 0.125, the endotoxin level of 3 cases of pigskin-derived gelatin was around 1.25, and the endotoxin level of high-grade gelatin from 3 cases was around 1.25. The endotoxin level in the example was less than 0.125. Endotoxin standard value of famotidine injection is 15 EU/mg, endotoxin standard value of thiamine chloride hydrochloride injection is 6.0 EU/mg, endotoxin standard value of roxatidine acetate hydrochloride for injection is 4.0 EU/mg, pyridoxine hydrochloride Considering that the endotoxin standard value for salt injection is 3.0 EU/mg and the endotoxin standard value for morphine hydrochloride injection is 1.5 EU/mg, endotoxin in tooth-derived collagen from 6 cases of Tokachi Wakagyu (registered trademark in Japan). It can be said that the endotoxin levels of the three cases of high-grade gelatin are extremely high, and the endotoxin levels of the three cases of pigskin-derived gelatin are also excellent.

In other words, the collagen-rich organic composition according to the present invention has a considerably reduced amount of endotoxin, or a considerably reduced activity of the endotoxin, and is substantially free of endotoxin or contains no endotoxin. It became clear that there was no endotoxin, or that the endotoxin contained was substantially inactivated, or that the endotoxin was inactivated.

Claims

A collagen-rich organic composition manufactured from teeth or bones extracted from a mammal, which is the following (i) or (i) and (ii):
(i) The amount of pyridinoline contained in collagen contained in the collagen-rich organic composition (ng/ PYD, which is a quantitative value of mg), is PYD>100,
(ii) The amount of pyridinoline contained in the collagen contained in the collagen-rich organic composition (ng/ PpP (contained pyridinoline amount (ng/mg)/contained pentosidine amount (ng/mg) ), PpP>740.
The collagen-rich organic material according to claim 1, which does not substantially contain endotoxin, or contains endotoxin, or contains substantially inactivated endotoxin, or has endotoxin inactivated. Composition.
The collagen-rich organic composition according to claim 1, wherein the mammal is one or more mammals selected from the group consisting of cows, pigs, horses, sheep, deer, dogs, cats, and humans.
A biomaterial made from the collagen-rich organic composition according to any one of claims 1 to 3, a biomaterial containing collagen as a main component, or other products.
A step of crushing a tooth or bone extracted from a mammal to obtain a crushed product;
a step of treating the obtained pulverized material with a strong alkali;
The method for producing a collagen-rich organic composition according to any one of claims 1 to 3, comprising a step of decalcifying the pulverized product subjected to the strong alkali treatment under negative pressure conditions.
According to claim 5, the step of treating the obtained pulverized material with a strong alkali is a step of treating the obtained pulverized material with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water. manufacturing method.
Quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in teeth or bones extracted from a mammal;
Quantifying the amount of pentosidine (ng/mg) contained in collagen contained in teeth or bones extracted from the mammal;
PpP (the amount of pyridinoline contained (ng /mg)/amount of pentosidine contained (ng/mg));
PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) or PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) and PpP (the amount of pyridinoline contained (ng /mg)/pentosidine content (ng/mg)) is a predetermined value, the teeth or bones extracted from the mammal can be used as biomaterials, collagen-based biomaterials, or raw materials for other products. A method for identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a collagen-based biomaterial, or other products, comprising:
A step of crushing a tooth or bone extracted from a mammal to obtain a crushed product;
a step of treating the obtained pulverized material with a strong alkali;
Deashing the strong alkali-treated pulverized material under negative pressure conditions to obtain an organic composition with a high collagen content;
Quantifying the amount of pyridinoline contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition;
Quantifying the amount of pentosidine contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition;
PpP (the amount of pyridinoline contained (ng /mg)/amount of pentosidine contained (ng/mg));
PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) or PYD which is the value of the quantified amount of pyridinoline contained (ng/mg) and PpP (the amount of pyridinoline contained (ng /mg)/pentosidine content (ng/mg)) is a predetermined value, the teeth or bones extracted from the mammal can be used as biomaterials, collagen-based biomaterials, or raw materials for other products. A method for identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a collagen-based biomaterial, or other products, comprising:
A step of quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in the obtained organic composition with high collagen content, and the amount of pentosidine contained in collagen contained in the obtained organic composition with high collagen content (ng/mg) ), the content of collagen contained in the obtained collagen-rich organic composition is determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pairing reagents. A step of quantifying the amount of pyridinoline (ng/mg) and a step of quantifying the amount of pentosidine contained in collagen (ng/mg) contained in the obtained collagen-rich organic composition, and the predetermined value is 9. A method according to claim 7 or claim 8, wherein PYD>100 or PYD>100 and PpP>740, respectively.
As an indicator for evaluating the potential risk of disease in a mammal and/or whether the mammal is potentially healthy, a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, or A method using a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and the quantitative value of the amount of pentosidine contained. There it is,
Quantifying and quantifying the amount of pyridinoline (ng/mg) contained in collagen contained in the teeth of a mammal, which is the target of evaluating the potential risk of disease and/or whether or not the mammal is potentially healthy. Obtaining the value PYD;
A step of quantifying the amount of pentosidine (ng/mg) contained in the collagen contained in the teeth of the mammal to be evaluated to obtain a quantitative value of PEN;
A step of calculating a weight ratio value of PpP (amount of pyridinoline contained (ng/mg)/amount of pentosidine contained (ng/mg)) from the obtained PYD and the obtained PEN;
The amount of pyridinoline contained in collagen contained in the teeth of one or more mammals selected from a mammal not suffering from a disease, a mammal suffering from a disease, a healthy mammal, and an unhealthy mammal. (ng/mg) to obtain a reference quantitative value of R-PYD;
The amount of pentosidine contained in collagen contained in the teeth of one or more mammals selected from a mammal not suffering from a disease, a mammal suffering from a disease, a healthy mammal, and an unhealthy mammal. (ng/mg) to obtain R-PEN, which is a reference quantitative value;
From the obtained R-PYD and the obtained R-PEN, calculate the reference value of weight ratio R-PpP (contained pyridinoline amount (ng / mg) / contained amount of pentosidine (ng / mg)) The process of
Comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and the PpP and the R-PpP.
Quantifying the amount of pyridinoline contained (ng/mg) to obtain R-PYD, which is a reference quantitative value, and quantifying the amount of pentosidine contained (ng/mg), obtaining R-PEN, which is a reference quantitative value. The step is to quantify the amount of pyridinoline contained (ng/mg) using a high performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents to determine the reference quantitative value of R-PYD. and a step of quantifying the pentosidine content (ng/mg) to obtain R-PEN as a reference quantitative value, and comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD. The step of comparing the R-PYD and comparing the PpP and the R-PpP checks whether the PYD is PYD>100, or whether the PYD is PYD>100. and checking whether the PpP is PpP>740.